For convenience, it is defined that analysis sensitivity of enzyme activity is the slope for the linear response of absorbance change rates to enzyme quantities, the limit of quantification (LOQ) is the quantity of an enzyme corresponding to the intercept of the linear response plus five times the standard error of estimate for the linear response. The assay of enzyme activity is always required to have an analysis sensitivity as high as possible, a LOQ as low as possible and analysis efficiency as high as possible. The use of specific chromogenic substrate of an enzyme facilitates the conventional and sensitive assay of enzyme activity via the continuous monitoring of reaction curves as absorbance changes; such a process for enzyme activity assay is widely utilized in conventional clinical biochemical analysis, health laboratory analysis to detect food contaminants and environment pollutants as enzyme inhibitors, high-throughput screening of enzyme inhibitor libraries and enzyme-labeled immunoassays. On the other hand, the high specificity and sensitivity for enzyme activity assay makes enzymes suitable labels for immunoassays represented by Enzyme-linked immunoassay (ELISA), which is the fundamental analysis method in clinical laboratory analysis of specific small biochemicals or macromolecules in mixture samples. During ELISA analysis, a component for capturing is immobilized in microplate wells to facilitate the separation of immunocomplexes, and wash process is needed; a single complete analysis process usually spends about 130 min or more, indicating low analysis efficiency. Therefore, the enhancement of the analysis efficiency of enzyme activity assay has great significance.
To date, continuous monitoring of reaction curves for enzyme activity assay always uses one substrate to measure the activity of just one enzyme each time, i.e., one enzyme in one reaction channel (solution). In clinical laboratory analysis, it is common to measure activities of two or even more enzymes in one sample, for instance, the diagnosis of liver function requires the assay of glutamic acid pyruvate aminotransferase, glutamic acid oxaloacetate aminotransferase, gamma-glutaminyltransferase (GGT), alkaline phosphatase, and so on in the same specimen; the diagnosis of acute pancreatitis requires the assay amylase and lipase in the same specimen. On the other hand, ELISA has a definite disadvantage of low efficiency since each analysis process needs a long time. Furthermore, the screening of libraries of enzyme inhibitors always determines the activities of just one target enzyme each time, resulting in high cost and low efficiency, and the consumption of more compounds in the libraries. The development of new techniques for enhancing the analysis efficiency of enzyme activity assay at sensitivity and LOQ comparable to those for separate assay of single component has its own biomedicinal significance.
The direct forward strategy to enhance the efficiency for enzyme activity assay is the real-time or nearly-real-time determination of the activities of multiple enzymes each time in one reaction channel by using multiple chromogenic substrates to continuous monitor absorbance changes of multiple chromogenic products. Spectrophotometers for concomitantly measuring multiple wavelength absorbances are already accessible, for example, Biotek ELX 800 microplate reader can concomitantly measure three wavelength absorbances, MAPADA UV 1600PC spectrophotometer can concomitantly measure seven wavelength absorbances. In fact, there are short enough lagging times between the assays of absorbance at different wavelengths with such instruments. When double monochromators or diode array detectors are used, there can be real-time simultaneous assays of absorbance at multiple wavelengths. Therefore, the development of principles for the combination of chromogenic substrates and techniques for data processing can potentially realize simultaneous measurement of the activities of multiple kinds of enzymes in single channel through the concomitant monitoring of multiple wavelength absorbances to obtain reaction processes of multiple enzymes, which can have sensitivity and LOQ for simultaneous measurement of the activities of multiple kinds of enzymes comparable to those for separate assays, and greatly accelerates ELISA and high-content screening of libraries of enzyme inhibitors.
It had long been reported for the use of two label enzymes for simultaneous ELISAs of two components in one reaction channel, but there were in fact no initiations of the reactions of two enzymes in the same reaction channel and continuous monitoring of the reaction processes of two enzymes. Blake et al in 1982 reported the use of calf intestinal alkaline phosphatase (CIAP) and β-galactosidase (β-gal) to label two haptens for simultaneous competitive assays of two free haptens in single reaction channel (Clin Chem 1982; 28(7):1469-1473). However, the reactions of such two labelled enzymes were initiated in two different reaction solutions rather than concomitantly in the same reaction solution, and the reaction processes of such two labelled enzymes were not monitored concurrently; this method used phenolphthalein monophosphate as the substrate of CIAP that had specific activity just about 7% of that on p-nitrophenylphosphate.
Too lower sensitivity and high cost on substrate made this improvement seldom practiced in biomedicine. In other ELISA techniques for the assays of two components in single reaction channel, there were no concomitant initiation of the reactions of two enzymes, no simultaneous assays of absorbance of two products and no continuous monitoring of reaction processes of two enzymes (Dean et al., Clin Chem, 1983; 29(6):1051-1056. Ng, et al. Clin Chem, 1987; 33(12): 2286-2288. Choi, et al. Clin Chem, 1991; 37(5): 673-677. Porstmann T, et al. J Immunol Methods. 1993; 158:95-106. Krambovitis E, et al. Clin chem 1995; 41:48-53. Osuchowski, et al. Methods 2006; 38:304-311 and Sun J, et al. Anal Chim Acta 2010; 666: 76-82). Therefore, to date, all ELISA techniques have not been improved to concomitantly initiate reactions of multiple label enzymes and concurrently monitor absorbance changes of multiple products as the reaction curves; in the field for enzyme inhibitor screening, there are no reports on simultaneous assay activities of multiple enzymes to detect the actions of one inhibitor on multiple target enzymes.
Clearly, the principles for the combination of chromogenic substrates and the techniques for data processing are required to develop a method for simultaneously measuring the activities of multiple kinds of enzymes through the concomitantly monitoring of multiple wavelength absorbances. It was under the support from “863”-program 2011AA02A108, the inventors developed the as-proposed novel method.